A Contaminant Impurity, Not Rigosertib, Is a Tubulin Binding Agent.

Rigosertib is a styryl benzyl sulfone that inhibits growth of tumor cells and acts as a RAS mimetic by binding to Ras binding domains of RAS effectors. A recent study attributed rigosertib's mechanism of action to microtubule binding. In that study, rigosertib was obtained from a commercial vendor. We compared the purity of clinical-grade and commercially sourced rigosertib and found that commercially sourced rigosertib contains approximately 5% ON01500, a potent inhibitor of tubulin polymerization. Clinical-grade rigosertib, which is free of this impurity, does not exhibit tubulin-binding activity. Cell lines expressing mutant ß-tubulin have also been reported to be resistant to rigosertib. However, our study showed that these cells failed to proliferate in the presence of rigosertib at concentrations that are lethal to wild-type cells. Rigosertib induced a senescence-like phenotype in the small percentage of surviving cells, which could be incorrectly scored as resistant using short-term cultures.

Discovery of 2-(1H-indol-5-ylamino)-6-(2,4-difluorophenylsulfonyl)-8-methylpyrido[2,3-d]pyrimidin-7(8H)-one (7ao) as a potent selective inhibitor of Polo like kinase 2 (PLK2).

Several families of protein kinases have been shown to play a critical role in the regulation of cell cycle progression, particularly progression through mitosis. These kinase families include the Aurora kinases, the Mps1 gene product and the Polo Like family of protein kinases (PLKs). The PLK family consists of five members and of these, the role of PLK1 in human cancer is well documented. PLK2 (SNK), which is highly homologous to PLK1, has been shown to play a critical role in centriole duplication and is also believed to play a regulatory role in the survival pathway by physically stabilizing the TSC1/2 complex in tumor cells under hypoxic conditions. As a part of our research program, we have developed a library of novel ATP mimetic chemotypes that are cytotoxic against a panel of cancer cell lines. We show that one of these chemotypes, the 6-arylsulfonyl pyridopyrimidinones, induces apoptosis of human tumor cell lines in nanomolar concentrations. The most potent of these compounds, 7ao, was found to be a highly specific inhibitor of PLK2 when profiled against a panel of 288 wild type, 55 mutant and 12 lipid kinases. Here, we describe the synthesis, structure activity relationship, in vitro kinase specificity and biological activity of the lead compound, 7ao.

Structural basis for the suppression of skin cancers by DNA polymerase eta.

DNA polymerase eta (Poleta) is unique among eukaryotic polymerases in its proficient ability for error-free replication through ultraviolet-induced cyclobutane pyrimidine dimers, and inactivation of Poleta (also known as POLH) in humans causes the variant form of xeroderma pigmentosum (XPV). We present the crystal structures of Saccharomyces cerevisiae Poleta (also known as RAD30) in ternary complex with a cis-syn thymine-thymine (T-T) dimer and with undamaged DNA. The structures reveal that the ability of Poleta to replicate efficiently through the ultraviolet-induced lesion derives from a simple and yet elegant mechanism, wherein the two Ts of the T-T dimer are accommodated in an active site cleft that is much more open than in other polymerases. We also show by structural, biochemical and genetic analysis that the two Ts are maintained in a stable configuration in the active site via interactions with Gln 55, Arg 73 and Met 74. Together, these features define the basis for Poleta's action on ultraviolet-damaged DNA that is crucial in suppressing the mutagenic and carcinogenic consequences of sun exposure, thereby reducing the incidence of skin cancers in humans.

Structure of the PAPP-ABP5 complex reveals mechanism of substrate recognition.

Insulin-like growth factor (IGF) signaling is highly conserved and tightly regulated by proteases including Pregnancy-Associated Plasma Protein A (PAPP-A). PAPP-A and its paralog PAPP-A2 are metalloproteases that mediate IGF bioavailability through cleavage of IGF binding proteins (IGFBPs). Here, we present single-particle cryo-EM structures of the catalytically inactive mutant PAPP-A (E483A) in complex with a peptide from its substrate IGFBP5 (PAPP-ABP5) and also in its substrate-free form, by leveraging the power of AlphaFold to generate a high quality predicted model as a starting template. We show that PAPP-A is a flexible trans-dimer that binds IGFBP5 via a 25-amino acid anchor peptide which extends into the metalloprotease active site. This unique IGFBP5 anchor peptide that mediates the specific PAPP-A-IGFBP5 interaction is not found in other PAPP-A substrates. Additionally, we illustrate the critical role of the PAPP-A central domain as it mediates both IGFBP5 recognition and trans-dimerization. We further demonstrate that PAPP-A trans-dimer formation and distal inter-domain interactions are both required for efficient proteolysis of IGFBP4, but dispensable for IGFBP5 cleavage. Together the structural and biochemical studies reveal the mechanism of PAPP-A substrate binding and selectivity.

Crystallization and preliminary crystallographic analysis of the type IIL restriction enzyme MmeI in complex with DNA.

Type IIL restriction enzymes have rejuvenated the search for user-specified DNA binding and cutting. By aligning and contrasting the highly comparable amino-acid sequences yet diverse recognition specificities across the family of enzymes, amino acids involved in DNA binding have been identified and mutated to produce alternative binding specificities. To date, the specificity of MmeI (a type IIL restriction enzyme) has successfully been altered at positions 3, 4 and 6 of the asymmetric TCCRAC (where R is a purine) DNA-recognition sequence. To further understand the structural basis of MmeI DNA-binding specificity, the enzyme has been crystallized in complex with its DNA substrate. The crystal belonged to space group P1, with unit-cell parameters a = 61.73, b = 94.96, c = 161.24 Å, α = 72.79, ß = 89.12, γ = 71.68°, and diffracted to 2.6 Å resolution when exposed to synchrotron radiation. The structure promises to reveal the basis of MmeI DNA-binding specificity and will complement efforts to create enzymes with novel specificities.

Sugar phosphate activation of the stress sensor eIF2B.

The multi-subunit translation initiation factor eIF2B is a control node for protein synthesis. eIF2B activity is canonically modulated through stress-responsive phosphorylation of its substrate eIF2. The eIF2B regulatory subcomplex is evolutionarily related to sugar-metabolizing enzymes, but the biological relevance of this relationship was unknown. To identify natural ligands that might regulate eIF2B, we conduct unbiased binding- and activity-based screens followed by structural studies. We find that sugar phosphates occupy the ancestral catalytic site in the eIF2Bα subunit, promote eIF2B holoenzyme formation and enhance enzymatic activity towards eIF2. A mutant in the eIF2Bα ligand pocket that causes Vanishing White Matter disease fails to engage and is not stimulated by sugar phosphates. These data underscore the importance of allosteric metabolite modulation for proper eIF2B function. We propose that eIF2B evolved to couple nutrient status via sugar phosphate sensing with the rate of protein synthesis, one of the most energetically costly cellular processes.

SOX11 Inhibitors Are Cytotoxic in Mantle Cell Lymphoma.

PURPOSE: Mantle cell lymphoma (MCL) is a fatal subtype of non-Hodgkin lymphoma. SOX11 transcription factor is overexpressed in the majority of nodal MCL. We have previously reported that B cell-specific overexpression of SOX11 promotes MCL pathogenesis via critically increasing BCR signaling in vivo. SOX11 is an attractive target for MCL therapy; however, no small-molecule inhibitor of SOX11 has been identified to date. Although transcription factors are generally considered undruggable, the ability of SOX11 to bind to the minor groove of DNA led us to hypothesize that there may exist cavities at the protein-DNA interface that are amenable to targeting by small molecules. EXPERIMENTAL DESIGN: Using a combination of in silico predictions and experimental validations, we report here the discovery of three structurally related compounds (SOX11i) that bind SOX11, perturb its interaction with DNA, and effect SOX11-specific anti-MCL cytotoxicity. RESULTS: We find mechanistic validation of on-target activity of these SOX11i in the inhibition of BCR signaling and the transcriptional modulation of SOX11 target genes, specifically, in SOX11-expressing MCL cells. One of the three SOX11i exhibits relatively superior in vitro activity and displays cytotoxic synergy with ibrutinib in SOX11-expressing MCL cells. Importantly, this SOX11i induces cytotoxicity specifically in SOX11-positive ibrutinib-resistant MCL patient samples and inhibits Bruton tyrosine kinase phosphorylation in a xenograft mouse model derived from one of these subjects. CONCLUSIONS: Taken together, our results provide a foundation for therapeutically targeting SOX11 in MCL by a novel class of small molecules.

A selective WDR5 degrader inhibits acute myeloid leukemia in patient-derived mouse models.

Interactions between WD40 repeat domain protein 5 (WDR5) and its various partners such as mixed lineage leukemia (MLL) and c-MYC are essential for sustaining oncogenesis in human cancers. However, inhibitors that block protein-protein interactions (PPIs) between WDR5 and its binding partners exhibit modest cancer cell killing effects and lack in vivo efficacy. Here, we present pharmacological degradation of WDR5 as a promising therapeutic strategy for treating WDR5-dependent tumors and report two high-resolution crystal structures of WDR5-degrader-E3 ligase ternary complexes. We identified an effective WDR5 degrader via structure-based design and demonstrated its in vitro and in vivo antitumor activities. On the basis of the crystal structure of an initial WDR5 degrader in complex with WDR5 and the E3 ligase von Hippel­Lindau (VHL), we designed a WDR5 degrader, MS67, and demonstrated the high cooperativity of MS67 binding to WDR5 and VHL by another ternary complex structure and biophysical characterization. MS67 potently and selectively depleted WDR5 and was more effective than WDR5 PPI inhibitors in suppressing transcription of WDR5-regulated genes, decreasing the chromatin-bound fraction of MLL complex components and c-MYC, and inhibiting the proliferation of cancer cells. In addition, MS67 suppressed malignant growth of MLL-rearranged acute myeloid leukemia patient cells in vitro and in vivo and was well tolerated in vivo. Collectively, our results demonstrate that structure-based design can be an effective strategy to identify highly active degraders and suggest that pharmacological degradation of WDR5 might be a promising treatment for WDR5-dependent cancers.

Structure and mechanism of B-family DNA polymerase ζ specialized for translesion DNA synthesis.

DNA polymerase ζ (Polζ) belongs to the same B-family as high-fidelity replicative polymerases, yet is specialized for the extension reaction in translesion DNA synthesis (TLS). Despite its importance in TLS, the structure of Polζ is unknown. We present cryo-EM structures of the Saccharomyces cerevisiae Polζ holoenzyme in the act of DNA synthesis (3.1 Å) and without DNA (4.1 Å). Polζ displays a pentameric ring-like architecture, with catalytic Rev3, accessory Pol31' Pol32 and two Rev7 subunits forming an uninterrupted daisy chain of protein-protein interactions. We also uncover the features that impose high fidelity during the nucleotide-incorporation step and those that accommodate mismatches and lesions during the extension reaction. Collectively, we decrypt the molecular underpinnings of Polζ's role in TLS and provide a framework for new cancer therapeutics.

JLP-centrosome is essential for the microtubule-mediated nucleocytoplasmic transport induced by extracellular stimuli.

JLP belongs to the JIP family whose members serve as scaffolding proteins that link motor proteins and their cargo for intracellular transport. Although JLP is mainly cytoplasmic, it accumulates as a focus in the perinuclear region when stimulated by extracellular stimuli. Focus formation, which changes the nucleus shape and concentrates the nuclear pores, depends on p38MAPK activation and the dynein retrograde motor protein complex. Extracellular stimuli trigger the tethering of PLK1 to the centrosome by JLP, leading to centrosome maturation and microtubule array formation. The centrosome localization domain of JLP is important for the binding of the centrosome and the formation of the JLP focus and the microtubule array. Furthermore, the formation of the JLP focus and the microtubule array is interdependent and important for the transport of NF-κB p65 to the nucleus and its unloading therein. In conclusion, JLP exhibits multiple functions in the nuclear translocation of NF-κB p65.

Cryo-EM structure and dynamics of eukaryotic DNA polymerase δ holoenzyme.

DNA polymerase δ (Polδ) plays pivotal roles in eukaryotic DNA replication and repair. Polδ is conserved from yeast to humans, and mutations in human Polδ have been implicated in various cancers. Saccharomyces cerevisiae Polδ consists of catalytic Pol3 and the regulatory Pol31 and Pol32 subunits. Here, we present the near atomic resolution (3.2 Å) cryo-EM structure of yeast Polδ holoenzyme in the act of DNA synthesis. The structure reveals an unexpected arrangement in which the regulatory subunits (Pol31 and Pol32) lie next to the exonuclease domain of Pol3 but do not engage the DNA. The Pol3 C-terminal domain contains a 4Fe-4S cluster and emerges as the keystone of Polδ assembly. We also show that the catalytic and regulatory subunits rotate relative to each other and that this is an intrinsic feature of the Polδ architecture. Collectively, the structure provides a framework for understanding DNA transactions at the replication fork.

Structure of Desulfitobacterium hafniense PylSc, a pyrrolysyl-tRNA synthetase.

Pyrrolysine, the 22nd genetically-encoded amino acid, is charged onto its specific tRNA by PylS, a pyrrolysyl-tRNA synthetase. While PylS is found as a single protein in certain archaeal methanogens, in the gram-positive bacterium Desulfitobacterium hafniense, PylS is divided into two separate proteins, PylSn and PylSc, corresponding to the N-terminal and C-terminal domains of the single PylS protein found in methanogens. Previous crystallographic studies have provided the structure of a truncated C-terminal portion of the archaeal Methanosarcina mazei PylS associated with catalysis. Here, we report the apo 2.1A resolution structure of the intact D. hafniense PylSc protein and compare it to structures of the C-terminal truncated PylS from methanogenic species. In PylSc, the hydrophobic pocket binding the ring of pyrrolysine is more constrained than in the archaeal enzyme; other structural differences are also apparent.

Crystallization and structure determination of the core-binding domain of bacteriophage lambda integrase.

Bacteriophage lambda integrase catalyzes site-specific DNA recombination. A helical bundle domain in the enzyme, called the core-binding domain (Int(CB)), promotes the catalysis of an intermediate DNA-cleavage reaction that is critical for recombination and is not well folded in solution in the absence of DNA. To gain structural insights into the mechanism behind the accessory role of this domain in catalysis, an attempt was made to crystallize an Int(CB)-DNA complex, but crystals of free Int(CB) were fortuitously obtained. The three-dimensional structure of DNA-free Int(CB) was solved at 2.0 A resolution by molecular replacement using as the search model the previously available DNA-bound 2.8 A structure of the Int(CB) domain in a larger construct of lambda integrase. The crystal structure of DNA-free Int(CB) resembles the DNA-bound structure of Int(CB), but exhibits subtle differences in the DNA-binding face and lacks electron density for ten residues in the C-terminus that form a portion of a linker connecting Int(CB) to the C-terminal catalytic domain of the enzyme. Thus, this work reveals the domain in the absence of DNA and allows comparison with the DNA-bound form of this catalytically activating domain.

Eukaryotic DNA polymerases.

The eukaryotic DNA replication machinery is conserved from yeast to humans and requires the actions of multiple DNA polymerases. In addition to replicative DNA polymerases for duplication of the leading and lagging DNA strands, another group of specialized polymerases is required for DNA repair and/or translesion DNA synthesis (TLS). We emphasize here recent findings that accelerate our understanding of the structure and mechanisms of these remarkable enzymes. We also highlight growing evidence on the role of DNA polymerases in the origin of certain cancers, and paradoxically as emerging targets for cancer therapy.

Metastatic cancers promote cachexia through ZIP14 upregulation in skeletal muscle.

Patients with metastatic cancer experience a severe loss of skeletal muscle mass and function known as cachexia. Cachexia is associated with poor prognosis and accelerated death in patients with cancer, yet its underlying mechanisms remain poorly understood. Here, we identify the metal-ion transporter ZRT- and IRT-like protein 14 (ZIP14) as a critical mediator of cancer-induced cachexia. ZIP14 is upregulated in cachectic muscles of mice and in patients with metastatic cancer and can be induced by TNF-α and TGF-ß cytokines. Strikingly, germline ablation or muscle-specific depletion of Zip14 markedly reduces muscle atrophy in metastatic cancer models. We find that ZIP14-mediated zinc uptake in muscle progenitor cells represses the expression of MyoD and Mef2c and blocks muscle-cell differentiation. Importantly, ZIP14-mediated zinc accumulation in differentiated muscle cells induces myosin heavy chain loss. These results highlight a previously unrecognized role for altered zinc homeostasis in metastatic cancer-induced muscle wasting and implicate ZIP14 as a therapeutic target for its treatment.

Development of a S-adenosylmethionine analog that intrudes the RNA-cap binding site of Zika methyltransferase.

The Zika virus (ZIKV) has emerged as a major health hazard. We present here a high resolution structure (1.55 Å) of ZIKV NS5 methyltransferase bound to a novel S-adenosylmethionine (SAM) analog in which a 4-fluorophenyl moiety substitutes for the methyl group. We show that the 4-fluorophenyl moiety extends into a portion of the RNA binding tunnel that typically contains the adenosine 2'OH of the RNA-cap moiety. Together, the new SAM analog and the high-resolution crystal structure are a step towards the development of antivirals against ZIKV and other flaviviruses.

Mechanism of error-free DNA synthesis across N1-methyl-deoxyadenosine by human DNA polymerase-ι.

N1-methyl-deoxyadenosine (1-MeA) is formed by methylation of deoxyadenosine at the N1 atom. 1-MeA presents a block to replicative DNA polymerases due to its inability to participate in Watson-Crick (W-C) base pairing. Here we determine how human DNA polymerase-ι (Polι) promotes error-free replication across 1-MeA. Steady state kinetic analyses indicate that Polι is ~100 fold more efficient in incorporating the correct nucleotide T versus the incorrect nucleotide C opposite 1-MeA. To understand the basis of this selectivity, we determined ternary structures of Polι bound to template 1-MeA and incoming dTTP or dCTP. In both structures, template 1-MeA rotates to the syn conformation but pairs differently with dTTP versus dCTP. Thus, whereas dTTP partakes in stable Hoogsteen base pairing with 1-MeA, dCTP fails to gain a "foothold" and is largely disordered. Together, our kinetic and structural studies show how Polι maintains discrimination between correct and incorrect incoming nucleotide opposite 1-MeA in preserving genome integrity.

Structure of the NS3 helicase from Zika virus.

Zika virus has emerged as a pathogen of major health concern. Here, we present a high-resolution (1.62-Å) crystal structure of the RNA helicase from the French Polynesia strain. The structure is similar to that of the RNA helicase from Dengue virus, with variability in the conformations of loops typically involved in binding ATP and RNA. We identify druggable 'hotspots' that are well suited for in silico and/or fragment-based high-throughput drug discovery.

Structures of NS5 Methyltransferase from Zika Virus.

The Zika virus (ZIKV) poses a major public health emergency. To aid in the development of antivirals, we present two high-resolution crystal structures of the ZIKV NS5 methyltransferase: one bound to S-adenosylmethionine (SAM) and the other bound to SAM and 7-methyl guanosine diphosphate (7-MeGpp). We identify features of ZIKV NS5 methyltransferase that lend to structure-based antiviral drug discovery. Specifically, SAM analogs with functionalities on the Cß atom of the methionine portion of the molecules that occupy the RNA binding tunnel may provide better specificity relative to human RNA methyltransferases.